Bacterial infections are of substantial global concern due to increasing antibiotic resistance. Staphylococcus aureus is a pathogen of alarm because of the ability of the bacterium to infect nearly every site in the human body. This adaptability combined with increasing antibiotic resistance results in the high levels of morbidity and mortality associated with staphylococcal infections. A powerful strategy utilized by vertebrates to combat bacterial pathogens is the sequestration of essential nutrients, a process known as nutritional immunity. Recent work found that during invasive staphylococcal disease mature abscesses are virtually devoid of manganese (Mn) and zinc (Zn). Further work revealed that the sequestration of Mn during staphylococcal infection is dependent on the neutrophil protein calprotectin (CP), a heterodimer of S100A8 and S100A9. CP is capable of binding one Mn ion and two Zn ions with nanomolar affinity, suggesting a role not only in Mn withholding but also in Zn sequestration. In a murine model of infection, CP-deficient mice have increased staphylococcal burdens, indicating that S. aureus is Mn starved during infection. This idea is further supported by the observation that during infection Mn-dependent bacterial processes are inhibited. In total, these results suggest that vertebrates cause S. aureus to become Mn and Zn starved during infection and that this starvation is critical to host defense. Even though vertebrates create an environment virtually devoid of Mn and Zn, S. aureus remains capable of causing significant and devastating disease. These observations led to the hypothesis that S. aureus must somehow adapt to the Mn and Zn limitation imposed by the host. S. aureus is known to encode dedicated Mn and Zn transporters that may help this pathogen resist the effects of vertebrate metal sequestration. However, the increased staphylococcal virulence in CP-deficient mice suggests that expression of Mn transporters, and presumably Zn transporters, by S. aureus is not sufficient to prevent metal starvation. Although it is known that vertebrates sequester Mn and Zn, the kinetics of this process have not been elucidated, and the extent of metal starvation imposed on S. aureus remain unknown. Furthermore, how S. aureus resists and adapts to Mn and Zn starvation imposed by CP remains unclear. Current projects are focused on elucidating the timing of Mn and Zn sequestration, the bacterial processes that are disrupted by this host defense, and the contribution of dedicated Mn and Zn import systems to resisting CP. Once a faculty position has been obtained, investigations will focus on determining how S. aureus adapts to Mn and Zn starvation. The work proposed in this application will follow up on the observations that the ArlRS two-component regulatory system and the two Mn-dependent superoxide dismutases expressed by S. aureus promote resistance to host imposed Mn and Zn limitation. The Specific Aims of this proposal are as follows: Aim I. Evaluate the role of ArlRS regulation and alterations in carbon source preference to facilitating S. aureus adaptation to host-imposed Mn and Zn limitation. Aim II. Determine the individual roles of S. aureus superoxide dismutases in resisting host-imposed metal starvation. In addition to providing substantial insight into the mechanisms that S. aureus uses to evade an important host defense, this proposal will facilitate the development of my independent research career. My long-term goal is to understand how the interactions between host and pathogen influence disease development. In pursuit of this goal, I obtained my graduate degree from Washington University in St. Louis with an emphasis in Molecular Microbiology and Microbial Pathogenesis followed by postdoctoral training in staphylococcal pathogenesis and metals biology with Dr. Eric Skaar at Vanderbilt University. My immediate goal is to obtain a faculty position and establish a highly productive independent research program. The commitment of Vanderbilt University to postdoctoral training, which includes offering seminars on how to run a successful independent laboratory, has substantially enhanced my ability to achieve this goal. To promote a successful transition from postdoctoral fellow to independent investigator, I will seek out advice and counsel from senior scientists at my future institution and an established network of informal advisors. I will also actively pursue opportunities to develop skills that will facilitate running a successful independent laboratory, and cultivate new collaborations. In combination, the proposed investigations and career development activities will provide an excellent foundation for the development of an independent research program.